During the past year, research in the Molecular Immunology Section has been focused on the mechanisms of recognition by human CD8+ T cell antigen-specific receptors (TCRs). Analysis of TCR recognition has been examined using T cell functional assays plus kinetic and thermodynamic binding assays, and crystallography. More recently, a variety of studies were designed to address one of the key questions in TCR biology: how does one TCR bind to multiple disparate ligands? The structural and biochemical basis of TCR cross-reactivity is still not well understood. In order to probe the mechanism of TCR cross-reactivity, CD8+ CTL were generated and characterized that are cross-reactive between a modified version of the HTLV-1 Tax 11-19 peptide coupled at position 5 to various haptens with significant differences in molecular structure. We completed the characterization of these TCRs with respect to their patterns of cross-reactivity and recognition of HLA-A2 molecules. In summary, these studies demonstrated that TCRs possess the molecular flexibility to accommodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule. Cloning and sequencing of a subset of these cross-reactive aa TCRs demonstrated that this high degree of molecular flexibility was not dependent on the utilization of unusually long CDR3 a or a chain loops.[unreadable] During the course of these studies on TCR cross-reactivity, it was discovered that the Tax 11-19-specific A6 TCR, which had been extensively characterized by our group in the past, is able to cross-react on the Tax peptide in which tyrosine 5 is replaced with a lysine conjugated to the hapten 4-(3-Indolyl)-butyric acid (Tax-5K-IBA). This A6 TCR/Tax-5K-IBA/HLA-A2 interface appears to be an extreme example of TCR cross-reactivity, since the presence of a 10-atom linker between the position 5 alpha carbon and the indole ring, as well as the differences between the substituted phenyl ring of tyrosine and the indole ring of IBA would be expected to impart significantly different chemical and structural features onto the peptide. Dr. Baker?s group performed A6 TCR/ligand binding studies and produced crystal structures of both the Tax-5K-IBA/HLA-A2 complex alone and the tri-molecular complex of the A6 TCR bound to Tax-5K-IBA/HLA-A2. Taken together, these functional and structural data indicate that TCR recognition is being mediated by a novel mechanism involving cooperative conformational plasticity in both the TCR and the hapten-modified peptide. The A6 TCR binds the Tax-5K-IBA/HLA-A2 complex by ?squishing? the hapten-peptide ligand into a folded structure compared to the unbound ligand.